We propose an experimental program aimed at determining basic cellular/synaptic mechanisms of local synaptic circuit organization in mouse primary motor cortex (M1). We present a multidisciplinary approach using laser scanning photostimulation (LSPS), pair recording, and related techniques for quantitative analysis of neocortical synaptic circuits. Our guiding hypothesis, based on preliminary data, is that local circuits in M1 - unlike sensory cortex - are adapted for `top down' control of motor output signals, in the form of massively convergent excitatory circuits from upper layers (layer 2/3) onto deeper layers (layers 5A, 5B, 6), and that this descending projection is composed of parallel intracortical pathways that are functionally specialized to integrate synaptic signals for corticospinal, corticostriatal, and other major M1 outputs. Our specific aims, testing different aspects of this general hypothesis, are as follows. First, because cortical layering is a primary determinant of cortical `wiring', in brain slice experiments we will record individually from pyramidal neurons located in all cortical layers in M1, and map the laminar and horizontal sources of excitatory synaptic input. This unique connectivity matrix data set will allow us to determine (for the first time for any cortical area) the average overall excitatory circuit organization in terms of the laminar locations of its neurons. Second, because cortical layers contain functionally distinct subclasses of neurons, we will determine the local circuit organization of major M1 neuronal subclasses. We will use retrograde tracers to identify corticospinal, corticocortical, and corticostriatal neurons for LSPS analysis. We will extend this analysis to determine circuit phenotypes for genetically labeled subclasses as well. Third, because the specific circuits identified above are likely to be functionally specialized, we will analyze their synaptic physiology using pair recording methods to measure unitary connection properties, including the amplitude, time course, and short term plasticity of synaptic signals. We will extend this analysis to the level of single-synapse properties through a novel combination of LSPS mapping and strontium treatment to isolate uniquantal events. Fourth, we will develop and use random access photostimulation to examine the efficacy and timing of feedforward synaptic excitation and inhibition within the M1 local circuit. This will reveal mechanisms of synaptic integration and coincidence detection in identified M1 synaptic pathways. The results will provide radically new insights for understanding the synaptic organization of M1 in wild type mice, providing a quantitative, mechanistic framework for future investigations of synaptic circuit pathophysiology in epilepsy, paralysis, and other disorders of voluntary motor control. PUBLIC HEALTH RELEVANCE Voluntary movements depend on synaptic circuits in the motor area of the neocortex (cortical gray matter) in the cerebral hemispheres. Here we propose a systematic, quantitative experimental approach that will elucidate fundamental synaptic signaling mechanisms and pathways in mammalian motor neocortex at the cellular level. The results will provide a much needed quantitative framework for understanding cortical circuit pathophysiology in epilepsy, paralysis, and related motor disorders.